ABSTRACT
Dominant de novo mutations in the co-chaperone BAG3 cause a severe form of myofibrillar myopathy, exhibiting progressive muscle weakness, muscle structural failure, and protein aggregation. To elucidate the mechanism of disease in, and identify therapies for, BAG3 myofibrillar myopathy, we generated two zebrafish models, one conditionally expressing BAG3P209L and one with a nonsense mutation in bag3. While transgenic BAG3P209L-expressing fish display protein aggregation, modeling the early phase of the disease, bag3-/- fish exhibit exercise dependent fiber disintegration, and reduced swimming activity, consistent with later stages of the disease. Detailed characterization of the bag3-/- fish, revealed an impairment in macroautophagic/autophagic activity, a defect we confirmed in BAG3 patient samples. Taken together, our data highlights that while BAG3P209L expression is sufficient to promote protein aggregation, it is the loss of BAG3 due to its sequestration within aggregates, which results in impaired autophagic activity, and subsequent muscle weakness. We therefore screened autophagy-promoting compounds for their effectiveness at removing protein aggregates, identifying nine including metformin. Further evaluation demonstrated metformin is not only able to bring about the removal of protein aggregates in zebrafish and human myoblasts but is also able to rescue the fiber disintegration and swimming deficit observed in the bag3-/- fish. Therefore, repurposing metformin provides a promising therapy for BAG3 myopathy.Abbreviations:ACTN: actinin, alpha; BAG3: BAG cochaperone 3; CRYAB: crystallin alpha B; DES: desmin; DMSO: dimethyl sulfoxide; DNAJB6: DnaJ heat shock protein family (Hsp40) member B6; dpf: days post fertilization; eGFP: enhanced green fluorescent protein; FDA: Food and Drug Administration; FHL1: four and a half LIM domains 1; FLNC: filamin C; hpf: hours post-fertilization; HSPB8: heat shock protein family B [small] member 8; LDB3/ZASP: LIM domain binding 3; MYOT: myotilin; TTN: titin; WT: wild-type.
Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis Regulatory Proteins , Metformin , Myopathies, Structural, Congenital , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis Regulatory Proteins/metabolism , Autophagy , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins , LIM Domain Proteins , Metformin/pharmacology , Molecular Chaperones/metabolism , Muscle Proteins , Muscles/metabolism , Mutation , Myopathies, Structural, Congenital/genetics , Nerve Tissue Proteins/metabolism , Zebrafish/metabolism , Zebrafish ProteinsABSTRACT
The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called "shuttling." In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, and involves alterations of phagocyte mobility, intercellular tethering, and phagosome transfer. Shuttling kinetics were fungal-species-specific, implicating a fungal determinant. ß-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favorable macrophage intracellular niche. Shuttling is a frequent host-pathogen interaction contributing to fungal infection establishment patterns.
Subject(s)
Aspergillosis/immunology , Host-Pathogen Interactions , Macrophages/physiology , Neutrophils/physiology , beta-Glucans/immunology , Animals , Aspergillus fumigatus , Mice , Phagocytosis , Phagosomes , Spores, Fungal , Talaromyces , ZebrafishABSTRACT
Although respiratory syncytial virus (RSV) is responsible for more human deaths each year than influenza, its pathogenic mechanisms are poorly understood. Here high-resolution quantitative imaging, bioenergetics measurements and mitochondrial membrane potential- and redox-sensitive dyes are used to define RSV's impact on host mitochondria for the first time, delineating RSV-induced microtubule/dynein-dependent mitochondrial perinuclear clustering, and translocation towards the microtubule-organizing centre. These changes are concomitant with impaired mitochondrial respiration, loss of mitochondrial membrane potential and increased production of mitochondrial reactive oxygen species (ROS). Strikingly, agents that target microtubule integrity the dynein motor protein, or inhibit mitochondrial ROS production strongly suppresses RSV virus production, including in a mouse model with concomitantly reduced virus-induced lung inflammation. The results establish RSV's unique ability to co-opt host cell mitochondria to facilitate viral infection, revealing the RSV-mitochondrial interface for the first time as a viable target for therapeutic intervention.
Subject(s)
Host-Pathogen Interactions , Mitochondria/pathology , Respiratory Syncytial Viruses/growth & development , Virus Replication , A549 Cells , Animals , Disease Models, Animal , Dyneins/metabolism , Humans , Lung/pathology , Lung/virology , Mice , Microtubules/metabolism , Respiratory Syncytial Virus Infections/pathologyABSTRACT
The ß-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of â¼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Cell Membrane/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Multiprotein Complexes/metabolism , Bacterial Outer Membrane Proteins/chemistry , Detergents/pharmacology , Escherichia coli Proteins/chemistry , Protein Biosynthesis/drug effects , Protein Multimerization , Protein Structure, SecondaryABSTRACT
Oxidative stress arising from suboptimal placental function contributes to a multitude of pathologies in infants compromised by fetal growth restriction (FGR). FGR infants are at high risk for respiratory dysfunction after birth and poor long-term lung function. Our objective was to investigate the contribution of oxidative stress to adverse lung development and the effects of melatonin administration, a powerful antioxidant, on lung structure in FGR lambs. Placental insufficiency and FGR was surgically induced in 13 fetal sheep at â¼105 days of gestation by ligation of a single umbilical artery. Maternal intravenous melatonin infusion was commenced in seven of the ewes 4 h after surgery and continued until birth. Lambs delivered normally at term and lungs were collected 24 h after birth for histological assessment of lung structure and injury and compared with appropriately grown control lambs (n = 8). FGR fetuses were hypoxic and had lower glucose during gestation compared with controls. Melatonin administration prevented chronic hypoxia. Within the lung, FGR caused reduced secondary septal crest density and altered elastin deposition compared with controls. Melatonin administration had no effect on the changes to lung structure induced by FGR. We conclude that chronic FGR disrupts septation of the developing alveoli, which is not altered by melatonin administration. These findings suggest that oxidative stress is not the mechanism driving altered lung structure in FGR neonates. Melatonin administration did not prevent disrupted airway development but also had no apparent adverse effects on fetal lung development.NEW & NOTEWORTHY Fetal growth restriction (FGR) results in poor respiratory outcomes, which may be caused by oxidation in utero. We investigated the contribution of oxidative stress to adverse lung development and the effects of melatonin administration, a powerful antioxidant, on lung structure in FGR lambs. FGR disrupted septation of the developing alveoli, which is not altered by melatonin administration. Oxidative stress may not be the mechanism driving altered lung structure in FGR neonates.
Subject(s)
Antioxidants/administration & dosage , Fetal Growth Retardation/drug therapy , Lung/drug effects , Lung/embryology , Melatonin/administration & dosage , Animals , Animals, Newborn , Female , Fetal Growth Retardation/diagnosis , Lung/pathology , Pregnancy , Pulmonary Alveoli/drug effects , Pulmonary Alveoli/embryology , Pulmonary Alveoli/pathology , Sheep , Treatment OutcomeABSTRACT
Human pathogenic Legionella replicate in alveolar macrophages and cause a potentially lethal form of pneumonia known as Legionnaires' disease(1). Here, we have identified a host-directed therapeutic approach to eliminate intracellular Legionella infections. We demonstrate that the genetic deletion, or pharmacological inhibition, of the host cell pro-survival protein BCL-XL induces intrinsic apoptosis of macrophages infected with virulent Legionella strains, thereby abrogating Legionella replication. BCL-XL is essential for the survival of Legionella-infected macrophages due to bacterial inhibition of host-cell protein synthesis, resulting in reduced levels of the short-lived, related BCL-2 pro-survival family member, MCL-1. Consequently, a single dose of a BCL-XL-targeted BH3-mimetic therapy, or myeloid cell-restricted deletion of BCL-XL, limits Legionella replication and prevents lethal lung infections in mice. These results indicate that repurposing BH3-mimetic compounds, originally developed to induce cancer cell apoptosis, may have efficacy in treating Legionnaires' and other diseases caused by intracellular microbes.
Subject(s)
Apoptosis , Legionella/growth & development , Macrophages, Alveolar/microbiology , Macrophages, Alveolar/physiology , bcl-X Protein/antagonists & inhibitors , Animals , Disease Models, Animal , Gene Deletion , Legionellosis/prevention & control , Mice , bcl-X Protein/geneticsABSTRACT
Innate cellular immune responses are a critical first-line defense against invading bacterial pathogens. Leukocyte migration from the bloodstream to a site of infection is mediated by chemotactic factors that are often host-derived. More recently, there has been a greater appreciation of the importance of bacterial factors driving neutrophil movement during infection. Here, we describe the development of a zebrafish infection model to study Acinetobacter baumannii pathogenesis. By using isogenic A. baumannii mutants lacking expression of virulence effector proteins, we demonstrated that bacterial drivers of disease severity are conserved between zebrafish and mammals. By using transgenic zebrafish with fluorescent phagocytes, we showed that a mutation of an established A. baumannii global virulence regulator led to marked changes in neutrophil behavior involving rapid neutrophil influx to a localized site of infection, followed by prolonged neutrophil dwelling. This neutrophilic response augmented bacterial clearance and was secondary to an impaired A. baumannii phenylacetic acid catabolism pathway, which led to accumulation of phenylacetate. Purified phenylacetate was confirmed to be a neutrophil chemoattractant. These data identify a previously unknown mechanism of bacterial-guided neutrophil chemotaxis in vivo, providing insight into the role of bacterial metabolism in host innate immune evasion. Furthermore, the work provides a potentially new therapeutic paradigm of targeting a bacterial metabolic pathway to augment host innate immune responses and attenuate disease.
Subject(s)
Acinetobacter Infections/immunology , Acinetobacter baumannii/genetics , Bacterial Proteins/genetics , Chemotaxis/drug effects , Phenylacetates/metabolism , Transcription Factors/genetics , Virulence Factors/genetics , Acinetobacter Infections/microbiology , Acinetobacter Infections/pathology , Acinetobacter baumannii/immunology , Acinetobacter baumannii/metabolism , Acinetobacter baumannii/pathogenicity , Animals , Animals, Genetically Modified , Chemotaxis/immunology , Embryo, Nonmammalian , Female , Gene Expression , Immunity, Innate , Metabolic Networks and Pathways/genetics , Mice , Mice, Inbred BALB C , Neutrophil Infiltration , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/microbiology , Phenylacetates/pharmacology , Transcription Factors/deficiency , Virulence , Virulence Factors/deficiency , ZebrafishABSTRACT
Podocyte depletion is sufficient for the development of numerous glomerular diseases and can be absolute (loss of podocytes) or relative (reduced number of podocytes per volume of glomerulus). Commonly used methods to quantify podocyte depletion introduce bias, whereas gold standard stereologic methodologies are time consuming and impractical. We developed a novel approach for assessing podocyte depletion in whole glomeruli that combines immunofluorescence, optical clearing, confocal microscopy, and three-dimensional analysis. We validated this method in a transgenic mouse model of selective podocyte depletion, in which we determined dose-dependent alterations in several quantitative indices of podocyte depletion. This new approach provides a quantitative tool for the comprehensive and time-efficient analysis of podocyte depletion in whole glomeruli.
Subject(s)
Cell Count/methods , Cell Size , Kidney Glomerulus/cytology , Podocytes/cytology , Animals , Imaging, Three-Dimensional , MiceABSTRACT
Deciphering the molecular basis of leukocyte recruitment is critical to the understanding of inflammation. In this study, we investigated the contribution of the tetraspanin CD37 to this key process. CD37-deficient mice showed impaired neutrophil recruitment in a peritonitis model. Intravital microscopic analysis indicated that the absence of CD37 impaired the capacity of leukocytes to follow a CXCL1 chemotactic gradient accurately in the interstitium. Moreover, analysis of CXCL1-induced leukocyte-endothelial cell interactions in postcapillary venules revealed that CXCL1-induced neutrophil adhesion and transmigration were reduced in the absence of CD37, consistent with a reduced capacity to undergo ß2 integrin-dependent adhesion. This result was supported by in vitro flow chamber experiments that demonstrated an impairment in adhesion of CD37-deficient neutrophils to the ß2 integrin ligand, ICAM-1, despite the normal display of high-affinity ß2 integrins. Superresolution microscopic assessment of localization of CD37 and CD18 in ICAM-1-adherent neutrophils demonstrated that these molecules do not significantly cocluster in the cell membrane, arguing against the possibility that CD37 regulates ß2 integrin function via a direct molecular interaction. Moreover, CD37 ablation did not affect ß2 integrin clustering. In contrast, the absence of CD37 in neutrophils impaired actin polymerization, cell spreading and polarization, dysregulated Rac-1 activation, and accelerated ß2 integrin internalization. Together, these data indicate that CD37 promotes neutrophil adhesion and recruitment via the promotion of cytoskeletal function downstream of integrin-mediated adhesion.
Subject(s)
Actins/metabolism , Antigens, CD/metabolism , Antigens, Neoplasm/metabolism , Cytoskeleton/immunology , Neutrophils/physiology , Tetraspanins/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Antigens, CD/genetics , Antigens, Neoplasm/genetics , CD18 Antigens/metabolism , Cell Adhesion , Cell Movement/genetics , Cells, Cultured , Chemokine CXCL1/metabolism , Chemotaxis/genetics , Intercellular Adhesion Molecule-1/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Binding , Tetraspanins/genetics , rac1 GTP-Binding Protein/geneticsSubject(s)
Acanthoma/virology , Carcinoma in Situ/virology , Carcinoma, Squamous Cell/virology , Papillomaviridae/isolation & purification , Papillomavirus Infections/virology , Precancerous Conditions/virology , Skin Neoplasms/virology , Acanthoma/pathology , Carcinoma in Situ/pathology , Carcinoma, Squamous Cell/pathology , DNA, Viral/analysis , Humans , Papillomaviridae/genetics , Papillomavirus Infections/pathology , Precancerous Conditions/pathology , Skin Neoplasms/pathologyABSTRACT
Childhood onset basal cell carcinoma is uncommon. In addition to occurring in children with albinism, Bazex syndrome, basal cell carcinoma nevus syndrome, nevus sebaceus, radiotherapy-treated cancers, solid organ transplants, and xeroderma pigmentosum, childhood onset basal cell carcinoma has also occurred, albeit less commonly, de novo. We describe a boy with idiopathic childhood onset basal cell carcinoma. Previously published children with de novo basal cell carcinoma were collected from computerized medical literature search (PubMed) and citations from earlier reports. To our knowledge, childhood onset idiopathic basal cell carcinoma has been observed in a total of 107 children, including our patient. Tumors were most commonly located on the head (90%). The tumors are most frequently nodular in appearance (52%) and in histology (at least 17%); however, aggressive histologic variants were observed in 20% of tumors. Basal cell carcinoma in children may be associated with prior sun exposure. The most common treatment is excision, with or without using the Mohs technique. However, 15 of 85 children, nearly 20%, developed recurrent tumors during a follow-up period ranging from 4 months to 20 years.
Subject(s)
Carcinoma, Basal Cell/pathology , Nose Neoplasms/pathology , Skin Neoplasms/pathology , Carcinoma, Basal Cell/surgery , Child , Humans , Male , Mohs Surgery , Nose Neoplasms/surgery , Skin Neoplasms/surgery , Treatment OutcomeABSTRACT
BACKGROUND: The purse string suture can be used to provide primary closure for small skin defects or as a partial closure for larger round wounds. The size of the defect is reduced secondary to the tension placed on the suture, which uniformly advances the skin from the entire periphery of the wound. METHODS: We reviewed retrospectively the features of 98 consecutive patients for whom a total of 100 cuticular purse string sutures were used to partially close their postoperative surgical defects. The location and types of the tumors removed were also summarized. RESULTS: Postoperative wounds were created following Mohs' micrographic excision of nonmelanoma skin cancer (basal cell carcinoma, 44; squamous cell carcinoma, 25), wide local excision of melanoma (29), or conservative excision of benign cutaneous neoplasms (two). The incidence of purse string suture for partial closure of each tumor was 4.1% for basal cell carcinoma, 7.3% for squamous cell carcinoma, and 46.3% for melanoma. The tumors were equally distributed on the trunk, head and neck, and extremities; however, purse string closures for basal cell carcinomas were more frequent on the trunk, head, and neck, relative to squamous cell carcinomas and melanomas, which were more common on the extremities. Concurrent medical problems and/or the use of an agent with anticoagulant or antiplatelet effects were noted in more than 50% of patients. Absorbable material of thicker diameter was most frequently used for the suture, and the postoperative wound area decreased by 6-90% (mean, 60%) following purse string partial closure. The suture was usually removed after 3-4 weeks. Postoperative complications occurred in six patients: allergic contact dermatitis in two, wound infection in two, exuberant granulation tissue in one, and hypertrophic scar in one. All of the wounds healed completely with either a round or linear scar. CONCLUSION: The cuticular purse string suture is a rapid and simple procedure that provides complete or partial closure of round skin defects and excellent long-term cosmetic and functional results. This closure provides uniform tension to the wound, enhances hemostasis at the tissue edge, and significantly decreases the size of the defect. Partial wound closure with the purse string suture may be advantageous following the local excision of melanoma, either as definitive surgical wound management or as a temporary partial wound closure prior to subsequent complete repair of the surgical defect. The purse string suture is also useful following nonmelanoma skin cancer removal in patients who insist on maintaining an active lifestyle in the immediate postoperative period, who are receiving one or more systemic anticoagulant and/or antiplatelet agents, and who have large surgical wounds that would require either a skin graft or a local cutaneous flap in order to close the postoperative defect.
Subject(s)
Carcinoma, Basal Cell/surgery , Carcinoma, Squamous Cell/surgery , Melanoma/surgery , Skin Neoplasms/surgery , Suture Techniques , Adult , Aged , Aged, 80 and over , Cicatrix/pathology , Female , Humans , Male , Middle Aged , Mohs Surgery , Retrospective Studies , Sutures , Wound HealingSubject(s)
Alopecia Areata/diagnosis , Alopecia Areata/therapy , Adult , Alopecia Areata/etiology , Biopsy , Diagnosis, Differential , Female , Humans , Immunosuppressive Agents/therapeutic use , Minoxidil/therapeutic use , Nurse's Role , Patient Education as Topic , Risk Factors , Vasodilator Agents/therapeutic useABSTRACT
Clear cell squamous cell carcinoma in situ, also referred to as pagetoid or clear cell Bowen disease, is a rare pathologic variant of this neoplasm. It is characterized by neoplastic cells with clear or pale cytoplasm. An octogenarian husband and wife concurrently developed new facial skin lesions which demonstrated squamous cell carcinoma in situ consisting of cancer cells with clear cytoplasm. Cutaneous human papillomavirus (HPV) typing detected HPV Type 5 and HPV Type 21 in the tumors of the husband and wife, respectively. HPV is a potential etiologic factor in the oncogenesis of nonmelanoma skin cancer, and HPV DNA has been demonstrated in extragenital squamous cell carcinoma in situ. The detection of DNA from different HPV types in the tumors of our patients suggests that the concurrent occurrence of their skin cancers may have been coincidental. However, the presence of HPV DNA in their tumors introduces the possibility of a viral-associated oncogenesis for clear cell squamous cell carcinoma in situ.
Subject(s)
Bowen's Disease/virology , Carcinoma in Situ/virology , Facial Neoplasms/virology , Papillomaviridae/isolation & purification , Skin Neoplasms/virology , Aged, 80 and over , Bowen's Disease/pathology , Bowen's Disease/surgery , Carcinoma in Situ/pathology , Carcinoma in Situ/surgery , Cytoplasm/pathology , Facial Neoplasms/pathology , Facial Neoplasms/surgery , Female , Humans , Male , Skin Neoplasms/pathology , Skin Neoplasms/surgery , SpousesABSTRACT
The purse-string suture provides complete or partial closure of round postoperative skin defects. It is a rapid and simple procedure to perform. Tension placed on the suture uniformly advances the skin from the entire periphery of the wound, resulting in a significant reduction of the defect size and enhancement of hemostasis at the wound edge. The history, modifications of the technique, advantages, and potential complications of the purse-string suture are reviewed. It is not only useful following the removal of nonmelanoma skin cancer but also after the local excision of melanoma. In addition, this technique is especially suitable for the repair of round surgical wounds for patients who are unable to modify their active lifestyles during the week following surgery, individuals concurrently being treated with anticoagulants, antiplatelet agents or both, and people with extensive postoperative defects that would otherwise require either a skin graft or a large cutaneous flap. Typically, the site of the surgical wound following partial or complete closure with the purse-string suture demonstrates excellent long-term cosmetic and functional results.
Subject(s)
Skin Neoplasms/surgery , Suture Techniques , Carcinoma, Basal Cell/surgery , Humans , Male , Middle Aged , Wound HealingSubject(s)
Hematoma , Nail Diseases , Running/injuries , Bandages , Drainage , Hematoma/diagnosis , Hematoma/etiology , Hematoma/therapy , Humans , Male , Middle Aged , Nail Diseases/diagnosis , Nail Diseases/etiology , Nail Diseases/therapy , Patient Education as Topic , Punctures , Skin Care/nursingABSTRACT
Ulceration of the nose may be inadvertently induced by the patient. Although trigeminal trophic syndrome is an uncommon cause of chronic ulcers, healthcare providers should consider the possibility of this disorder when encountering a patient with nasal ulcerations. Trigeminal trophic syndrome most commonly occurs in older women following therapy for trigeminal neuralgia. The ulcers usually involve the nasal ala and paranasal areas. The clinical vignette of a man with a self-induced nasal ulcer secondary to trigeminal trophic syndrome, which was initially suspected to be skin cancer, is presented. Since nasal ulcerations can be secondary to other conditions, a lesional biopsy should be performed to exclude other diagnoses when trigeminal trophic syndrome is entertained. In addition to trigeminal trophic syndrome, the differential diagnosis of conditions that can cause nasal ulcers include factitial disorders with self-induced ulcerations (such as dermatitis artifacta and neurotic excoriations), granulomatous conditions, infectious diseases, malignancy, and pyoderma gangrenosum. Treatment of trigeminal trophic syndrome requires prevention of digital manipulation of the lesion-either by occluding contact with the ulcer, initiating psychotropic medication, or both. Psychiatric and/or pharmacologic intervention should be considered to reduce or resolve further habitual self-inflicted injury before surgical intervention.